PROJECT SUMMARY/ABSTRACT Oral antiretroviral therapy (ART) is highly effective for HIV-infected people to prevent transmission and HIV- uninfected people to prevent acquisition, known as pre-exposure prophylaxis (PrEP), but monitoring and improving drug adherence have been challenging. The World Health Organization recommends all HIV-infected people receive ART and all people at substantial risk of HIV infection receive PrEP with priority for high incidence groups (?3 infections/100 person-years) in both the US and developing countries. However, ART and PrEP are only effective when people maintain drug levels equivalent to ?4 doses per week. As many patients taking ART or PrEP have difficulty with adherence, clinicians currently have no available tools to monitor drug levels or adherence to provide targeted counseling and support. We propose that a simple, rapid, point-of-care assay to monitor ART or PrEP drug adherence could be a useful tool for clinicians and patients to monitor drug levels and guide adherence counseling, which may lead to less HIV transmission and better coordination of HIV prevention efforts. Since 2013, we have been developing a simple, point-of-care assay to accurately and inexpensively measure tenofovir, the primary drug of both ART and PrEP. In 2016, we initiated an NIH-funded pharmacokinetic study to establish the necessary limit of detection and drug levels for our point-of-care PrEP adherence test. Therefore, we will complete the development of the rapid tenofovir test, transfer the technology to a lateral flow point-of-care assay format with the appropriate detection threshold, and conduct a clinical feasibility, acceptability and validation study in a local PrEP clinic. Our objective in this application is to complete the development of a rapid lateral flow point-of-care test to monitor and improve adherence to ART and PrEP, and to assess feasibility, acceptability, and validation in a PrEP clinic. We plan to test our central hypotheses by pursuing the following three specific aims: (1) To optimize the detection and measurement of tenofovir in buffer, blood, and urine specimens using antibodies, aptamers, or nanobodies; (2) To transfer the immunoassay components into a lateral flow assay format and establish the quantitative range and limit of detection of tenofovir in blood and urine specimens; (3) To assess clinical feasibility and validation of the point-of-care tenofovir test among PrEP providers and patients in a clinical setting. This work is innovative because it develops an entirely new category of rapid diagnostic test for monitoring adherence, which will help patients and clinicians improve ART and PrEP adherence and prevent HIV transmission. At the end of this project, we expect to have a rapid lateral flow assay that will be suitable for a future large-scale clinical implementation trial. If our rapid tenofovir adherence test proves to be valuable and cost-effective, then it could be easily implemented in clinics worldwide, including resource-limited settings.